Over the last several years, a few drugs have been identified that inhibit the methylation or de-acetylation pathways mediated by DNA methyltransferases (DNMT) and histone deacetylases (HDAC). These compounds have had immediate application in the treatment of cancers because of their ability to reactivate aberrantly silenced tumor suppressor genes. The need to identify more small molecules that target these two enzyme families is clear and has been repeatedly stated in the field of cancer biology. Our overall goal is to expand the available repertoire of drugs that can modulate gene expression and to evaluate their basic mechanism of action. Toward this goal, we have recently developed a unique mammalian cell-based system to screen for such small molecules in a reliable, simple fashion that is amenable to automation. Unlike methods used to date, our system has the advantage of measuring the exact biological event it seeks to target: the reversal of transcriptional repression in mammalian cells. The assay has been tested in screens of small molecule libraries and has successfully resulted in eight hits. Preliminary evaluation of one hit compound and one analog has revealed HDAC inhibitor properties. Because these compounds are structurally distinct from known HDAC/DNMT inhibitors, they will contribute to improving the diversity of this drug type. The funding of this proposal will allow us to characterize the mechanism of action of the identified compounds and to evaluate their anti-cancer activity. Our specific aims are: 1: To measure the ability of the compounds identified in our high content screen to inhibit the enzymatic activity of HDAC or DNMT enzymes in vitro; 2: To evaluate the ability of the candidate epigenetic modulators to inhibit the growth of human cancer cells and to re-activate the expression of aberrantly silenced genes in human cancer cell lines; and 3: To measure the effects of these epigenetic modulators on tumor growth in xenograft models. These studies will provide the foundation for further work in the development of these potential cancer therapeutic compounds and allow us to move into pre-clinical drug evaluation. Public health relevance: We have developed a system to identify drugs with the ability to turn on genes that are abnormally turned off in cancer cells. Using this system, we have already identified eight candidate drugs and propose to characterize them measuring their ability to block the growth of human cancer cells.